This project is designed to generate databases of novel candidate transcription factors that influence maintenance of mammalian spermatogonial stem cells (SSCs) via self-renewal and activation of the differentiating transition that occurs in transit amplifying progenitor spermatogonia in response to retinoic acid (RA) stimulation. These activities are the foundation for continual spermatogenesis and hence male fertility. Achieving the goals of this project will provide new information that will inform the field to explore novel molecular networks that define the stem cell and progenitor states in the male germline. Capitalizing on the Id4-eGfp transgenic mouse line that we created in previous studies to distinguish the SSC and progenitor pools and the capacity to generate primary cultures of undifferentiated spermatogonia, we have devised a novel high throughput screening approach to explore the role of >1,400 transcription factors in regulating the fate decisions of these cells. Our specific aims are to: 1) perform a high throughput screen to identify novel transcription factors that influence maintenance of the SSC pool; and 2) perform a high throughput screen to identify novel transcriptions factors mediating the RA response in progenitor spermatogonia. The outcomes of this project are expected to provide invaluable databases for progressive research into molecular mechanisms governing the foundational processes of spermatogenesis in mammalian testes. The potential long-term impact is that the information will be used to uncover molecular deficiencies that cause idiopathic male azoospermic infertility, and the development of novel therapeutic strategies to address the pathology or protect the undifferentiated spermatogonial population from cancer therapies that often lead to depletion of the SSC pool and infertility.